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Retroviral Transduction of Murine and Human Hematopoietic Progenitors and Stem Cells

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Hematopoietic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1185))

Abstract

Genetic modification of cells using retroviral vectors is the method of choice when the cell population is difficult to transfect and/or requires persistent transgene expression in progeny cells. There are innumerable potential applications for these procedures in laboratory research and clinical therapeutic interventions. One paradigmatic example is the genetic modification of hematopoietic stem and progenitor cells (HSPCs). These are rare nucleated cells which reside in a specialized microenvironment within the bone marrow, and have the potential to self-renew and/or differentiate into all hematopoietic lineages. Due to their enormous regenerative capacity in steady state or under stress conditions these cells are routinely used in allogeneic bone marrow transplantation to reconstitute the hematopoietic system in patients with metabolic, inflammatory, malignant, and other hematologic disorders. For patients lacking a matched bone marrow donor, gene therapy of autologous hematopoietic stem cells has proven to be an alternative as highlighted recently by several successful gene therapy trials.

Genetic modification of HSPCs using retrovirus vectors requires ex vivo manipulation to efficiently introduce the new genetic material into cells (transduction). Optimal culture conditions are essential to facilitate this process while preserving the stemness of the cells. The most frequently used retroviral vector systems for the genetic modifications of HSPCs are derived either from Moloney murine leukemia-virus (Mo-MLV) or the human immunodeficiency virus-1 (HIV-1) and are generally termed according to their genus gamma-retroviral (γ-RV) or lentiviral vectors (LV), respectively. This chapter describes in a step-by-step fashion some techniques used to produce research grade vector supernatants and to obtain purified murine or human hematopoietic stem cells for transduction, as well as follow-up methods for analysis of transduced cell populations.

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Acknowledgments

This manuscript is dedicated to past and present members of the Williams’s laboratory, who have helped to develop many methods described herein. Thanks to Dr. Mathilde Gavillet, Jenna Wood for assistance and proofing the manuscript. This work was supported by NIH grants (R01 CA113969, R24 DK099808, and R01 DK062757) and the German Academic Exchange Service grant (DAAD D/12/03783).

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Authors and Affiliations

  1. Boston Children’s Hospital, Dana Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, 300 Longwood Ave., Karp 08125.3, 02115, Boston, MA, USA

    Marioara F. Ciuculescu, Christian Brendel, Chad E. Harris & David A. Williams

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  1. Marioara F. Ciuculescu
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  2. Christian Brendel
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  3. Chad E. Harris
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  4. David A. Williams
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Corresponding author

Correspondence to David A. Williams .

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Editors and Affiliations

  1. Department of Pediatrics, Aflac Cancer and Blood Disorders Center Emory University School of Medicine, Atlanta, Georgia, USA

    Kevin D. Bunting

  2. Department of Pediatrics, Emory University School of Medicine Aflac Cancer & Blood Disorders Ctr, Atlanta, Georgia, USA

    Cheng-Kui Qu

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Ciuculescu, M.F., Brendel, C., Harris, C.E., Williams, D.A. (2014). Retroviral Transduction of Murine and Human Hematopoietic Progenitors and Stem Cells. In: Bunting, K., Qu, CK. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Biology, vol 1185. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1133-2_20

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  • DOI: https://doi.org/10.1007/978-1-4939-1133-2_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1132-5

  • Online ISBN: 978-1-4939-1133-2

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